It is well known to perform a quantitative or qualitative analysis of an aqueous liquid by contacting that liquid with a combination of reagents capable of yielding a detectable product in proportion to the concentration of the analyte in the liquid. As used herein, this combination of reagents is termed an interactive composition which is capable of chemical reactivity, catalytic activity, or any other form of chemical or physical interaction that can result in the ultimate production of a change that is detectable with suitable procedures and equipment.
One type of useful assay utilizes enzymatic reactions wherein the analyte, upon contact with the appropriate reagents, reacts with oxygen in the presence of a suitable enzyme to produce hydrogen perozide in proportion to the concentration of the analyte. A detectable product is then produced by the reaction of hydrogen peroxide in proportion to the concentration of the analyte in the tested liquid. Peroxidase is generally used in such assays to catalyze the oxidation of interactive composition by hydrogen peroxide.
Peroxidase can be used for diagnostic determinations of various analytes such as glucose, triglycerides, uric acid, cholesterol, creatine kinase, etc. and can be used as a label in ligand analogs used in the determination of immunologically reactive species (i.e. immunoassays). Such determinations can be carried out in solution or in dry analytical elements, such as those described in U.S. Pat. Nos. 3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al), 4,089,747 (issued May 16, 1978 to Bruschi) and 4,258,001 (issued Mar. 24, 1981 to Pierce et al).
The rate of reaction of various substrates with peroxidase varies over many orders of magnitude. In some instances, where the reaction proceeds slowly, a large amount of peroxidase is used to increase the reaction rate. However, the use of large amounts of peroxidase to increase the rate of reaction cannot be used in certain assays. For example, enzyme immunoassays using a peroxidase-labeled ligand analog have become important for determining a drug, antigen or other immunologically reactive compound. In such assays, a large amount of peroxidase cannot be added to increase the enzymatic reaction rate.
E.P. Publication No. 116,454 (published Aug. 22, 1984) describes an immunoassay using peroxidase, an oxidant (e.g., hydrogen peroxide), a chemiluminescent substrate and a phenol as a sensitivity enhancer. These enhancers allegedly increase the sensitivity of the chemiluminescent assay thereby providing a higher quantity of measurable light in the assay. No leuco dyes are used in a chemiluminescent assay. There is no suggestion in this reference that certain phenols or anilines could act as electron transfer agents to increase the rate of oxidation of leuco dyes. Chemiluminescent assays have the disadvantages of (1) requiring specialized equipment, (2) being excessively sensitive to sample volume changes, (3) often requiring relatively high pH, and (4) exhibiting poor assay precision. Hence, there are sufficient reasons for avoiding chemiluminescent assays if possible.
Useful assays utilizing triarylimidazole leuco dyes are described in U.S. Pat. No. 4,089,747, noted above. However, it has been observed in such assays that the oxidation of the leuco dye by peroxidase is relatively slow. Large amounts of peroxidase are needed to increase the rate of reaction, but using increased amounts of peroxidase is often undesirable or impractical for economic reasons or because increased amounts adversely affect the assay, e.g. when peroxidase is used as a label in immunoassays. Hence, it would be desirable to be able to increase the reaction rate of peroxidase catalyzed oxidation of leuco dyes without having to increase the amount of peroxidase.